Adenoviral vector-based foot-and-mouth disease vaccine

ABSTRACT

The invention is directed to an adenoviral vector comprising at least one nucleic acid sequence encoding an  aphthovirus  antigen and/or a cytokine operably linked to a promoter. The adenoviral vector is replication-deficient and requires at most complementation of both the E1 region and the E4 region of the adenoviral genome for propagation. The invention also is directed to a method of inducing an immune response in a mammal comprising administering to the mammal a composition comprising the aforementioned adenoviral vector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of copending InternationalPatent Application No. PCT/US2006/060830, filed Nov. 13, 2006,designating the United States, which claims the benefit of U.S.Provisional Patent Application No. 60/735,439, filed Nov. 10, 2005.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Other TransactionAgreement (OTA) No. HSHQDC-07-9-00004 awarded by the United StatesDepartment of Homeland Security (DHS). The Government has certain rightsin this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 1,139 Byte ASCII (Text) file named“702949_ST25.TXT,” created on Jun. 23, 2009.

BACKGROUND OF THE INVENTION

Foot-and-mouth disease (FMD) is a highly contagious disease ofcloven-hooved animals, including cattle, swine, sheep, goats, and deer,that rapidly replicates in the host and spreads to susceptible animalsby contact or aerosol. Because of the highly infectious nature of FMD,countries free of the disease maintain rigid quarantine and importrestrictions on animals and animal products from infected countries inorder to prevent its introduction and to allow continued activeparticipation in international trade. The disease does not occur in theU.S., Canada, or Mexico, and its continued absence from North America isa priority for the U.S. livestock industry and the United StatesDepartment of Agriculture (USDA).

The virus that causes FMD (FMDV) is an RNA virus classified as a memberof the genus Aphthovirus and the family Picornaviridae (see Cooper etal., Intervirology, 10: 165-180 (1978)). There are seven known serotypesof FMDV: the European serotypes A, O and C, the South Africa Territoriesserotypes SAT 1, SAT 2, and SAT 3, and the Asia 1 serotype. A number ofantigenically distinct subtypes are recognized within each of theseserotypes. Indeed, for each serotype or subtype several geneticallydistinct variants exist.

Disease incidence in previously FMD-free countries, such as the UnitedKingdom in 2001 (see, e.g. Knowles et al., Vet. Rec., 148: 258-259(2001)) are controlled by inhibition of susceptible animal movement,slaughter of infected and in-contact animals, and decontamination.Inactivated whole virus vaccines are conventionally used in FMD controlprograms as a last resort mainly because of the adverse economic affectsof vaccination as compared to slaughter, despite their success incontrolling the disease. Other problems associated with the currentlyavailable FMD vaccine include a requirement for high-containmentfacilities to produce the virus needed for vaccine manufacture, theantigenic variation of the virus resulting in numerous virus serotypesand subtypes, and the inability of vaccines to rapidly induce protectiveimmunity.

To circumvent these problems, researchers have explored using viralvectors as FMD vaccines. For example, E1-deficient adenoviral vectorshave been engineered to encode the FMD virus (FMDV) empty capsid and the3C protease (Pacheco et al., Virology, 337: 205-209 (2005)), as well asinterferons (U.S. Patent Application Publication 2003/0171314 A1). Suchadenoviral vectors, however, have not been shown to induce the rapidantibody response required to combat an FMDV outbreak.

Accordingly, there remains a need for viral vector vaccines that elicita more rapid and complete immune response against foot-and-mouthdisease. The invention provides such viral vectors.

BRIEF SUMMARY OF THE INVENTION

The invention provides an adenoviral vector comprising an adenoviralgenome and at least one nucleic acid sequence encoding an aphthovirusantigen and/or a cytokine operably linked to a promoter, wherein theadenoviral vector is replication-deficient and requires complementationof both the E1 region and the E4 region of the adenoviral genome forpropagation.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1A is a table illustrating the results of a vaccination-challengestudy in cattle immunized with 5×10⁹ FFU of the adenoviral vector A24GV11. “VNT” denotes virus neutralization titer, “VI” denotes virusisolation, “GI” denotes generalization of infection, “F” denotes fever,“N” denotes negative, and “P” denotes positive.

FIG. 1B is a table illustrating the results of a vaccination-challengestudy in cattle immunized with 1×10⁸ FFU of the adenoviral vector A24GV11. “VNT” denotes virus neutralization titer, “VI” denotes virusisolation, “GI” denotes generalization of infection, “F” denotes fever,“N” denotes negative, and “P” denotes positive.

FIG. 1C is a table illustrating the results of a vaccination-challengestudy in cattle immunized with 5×10⁶ FFU of the adenoviral vector A24GV11. “VNT” denotes virus neutralization titer, “VI” denotes virusisolation, “GI” denotes generalization of infection, “F” denotes fever,“N” denotes negative, and “P” denotes positive.

FIG. 1D is a table illustrating the results of a vaccination-challengestudy in control cattle that were not immunized prior to FMDV challenge.“VNT” denotes virus neutralization titer, “VI” denotes virus isolation,“GI” denotes generalization of infection, “F” denotes fever, “N” denotesnegative, and “P” denotes positive.

FIG. 2A is a graph illustrating the neutralizing antibody responseagainst FMDV strain A24 in cattle produced as a result of thevaccination-challenge study described in Example 1. The antibody titerswere measured prior to vaccination (D0pv), seven days after vaccination(D7 pv/D0 pch), and 14 days post challenge (D14 pch).

FIG. 2B is a graph illustrating the neutralizing antibody responseagainst serotype 5 adenovirus in cattle produced as a result of thevaccination-challenge study described in Example 1. The antibody titerswere measured prior to vaccination (D0pv), seven days after vaccination(D7 pv/D0 pch), and 14 days post challenge (D14 pch).

FIG. 3A is a graph illustrating the neutralizing antibody responseagainst FMDV strain A24 in cattle produced as a result of thevaccination-challenge study described in Example 1. The antibody titerswere measured seven days prior to vaccination (D7), the day ofvaccination (D0), and 14 days post challenge (D14 pch).

FIG. 3B is a graph illustrating the neutralizing antibody responseagainst serotype 5 adenovirus in cattle produced as a result of thevaccination-challenge study described in Example 1. The antibody titerswere measured seven days prior to vaccination (D7), the day ofvaccination (D0), and 14 days post challenge (D14 pch).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an adenoviral vector comprising an adenoviralgenome comprising at least one nucleic acid sequence encoding anaphthovirus antigen and/or a cytokine operably linked to a promoter,wherein the adenoviral vector is replication-deficient and requires atmost complementation of both the E1 region and the E4 region of theadenoviral genome for propagation. Adenovirus (Ad) is a 36 kbdouble-stranded DNA virus that efficiently transfers DNA in vivo to avariety of different target cell types. For use in the invention, theadenovirus is preferably made replication deficient by deleting, inwhole or in part, select genes required for viral replication. Theexpendable E3 region is also frequently deleted, in whole or in part, toallow additional room for a larger DNA insert. The vector can beproduced in high titers and can efficiently transfer DNA to replicatingand non-replicating cells. The newly transferred genetic informationremains epi-chromosomal, thus eliminating the risks of randominsertional mutagenesis and permanent alteration of the genotype of thetarget cell. However, if desired, the integrative properties of AAV canbe conferred to adenovirus by constructing an AAV-Ad chimeric vector.For example, the AAV ITRs and nucleic acid encoding the Rep proteinincorporated into an adenoviral vector enables the adenoviral vector tointegrate into a mammalian cell genome. Therefore, AAV-Ad chimericvectors can be a desirable option for use in the invention.

Adenovirus from various origins, subtypes, or mixture of subtypes can beused as the source of the viral genome for the adenoviral vector. Whilenon-human adenovirus (e.g., simian, avian, canine, ovine, or bovineadenoviruses) can be used to generate the adenoviral vector, a humanadenovirus preferably is used as the source of the viral genome for theadenoviral vector. For instance, an adenovirus can be of subgroup A(e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11,14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32,33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g.,serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and51), or any other adenoviral serotype. Adenoviral serotypes 1 through 51are available from the American Type Culture Collection (ATCC, Manassas,Va.). Preferably, in the context of the invention, the adenoviral vectoris of human subgroup C, especially serotype 2 or even more desirablyserotype 5. However, non-group C adenoviruses can be used to prepareadenoviral gene transfer vectors for delivery of gene products to hostcells. Preferred adenoviruses used in the construction of non-group Cadenoviral gene transfer vectors include Ad12 (group A), Ad7 and Ad35(group B), Ad30 and Ad36 (group D), Ad4 (group E), and Ad41 (group F).Non-group C adenoviral vectors, methods of producing non-group Cadenoviral vectors, and methods of using non-group C adenoviral vectorsare disclosed in, for example, U.S. Pat. Nos. 5,801,030, 5,837,511, and5,849,561 and International Patent Applications WO 97/12986 and WO98/53087.

The adenoviral vector can comprise a mixture of subtypes and thereby bea “chimeric” adenoviral vector. A chimeric adenoviral vector cancomprise an adenoviral genome that is derived from two or more (e.g., 2,3, 4, etc.) different adenovirus serotypes. In the context of theinvention, a chimeric adenoviral vector can comprise different orapproximately equal amounts of the genome of each of the two or moredifferent adenovirus serotypes. When the chimeric adenoviral vectorgenome is comprised of the genomes of two different adenovirusserotypes, the chimeric adenoviral vector genome preferably comprises nomore than about 70% (e.g., no more than about 65%, about 50%, or about40%) of the genome of one of the adenovirus serotypes, with theremainder of the chimeric adenovirus genome being derived from thegenome of the other adenovirus serotype. In one embodiment, the chimericadenoviral vector can contain an adenoviral genome comprising a portionof a serotype 2 genome and a portion of a serotype 5 genome. Forexample, nucleotides 1-456 of such an adenoviral vector can be derivedfrom a serotype 2 genome, while the remainder of the adenoviral genomecan be derived from a serotype 5 genome.

The adenoviral vector of the invention can be replication-competent. Forexample, the adenoviral vector can have a mutation (e.g., a deletion, aninsertion, or a substitution) in the adenoviral genome that does notinhibit viral replication in host cells. The adenoviral vector also canbe conditionally replication-competent. Preferably, however, theadenoviral vector is replication-deficient in host cells.

By “replication-deficient” is meant that the adenoviral vector requirescomplementation of one or more regions of the adenoviral genome that arerequired for replication, as a result of, for example, a deficiency inat least one replication-essential gene function (i.e., such that theadenoviral vector does not replicate in typical host cells, especiallythose in an animal that could be infected by the adenoviral vector inthe course of the inventive method). A deficiency in a gene, genefunction, gene, or genomic region, as used herein, is defined as amutation or deletion of sufficient genetic material of the viral genometo obliterate or impair the function of the gene (e.g., such that thefunction of the gene product is reduced by at least about 2-fold,5-fold, 10-fold, 20-fold, 30-fold, or 50-fold) whose nucleic acidsequence was mutated or deleted in whole or in part. Deletion of anentire gene region often is not required for disruption of areplication-essential gene function. However, for the purpose ofproviding sufficient space in the adenoviral genome for one or moretransgenes, removal of a majority of a gene region may be desirable.While deletion of genetic material is preferred, mutation of geneticmaterial by addition or substitution also is appropriate for disruptinggene function. Replication-essential gene functions are those genefunctions that are required for replication (e.g., propagation) and areencoded by, for example, the adenoviral early regions (e.g., the E1, E2,and E4 regions), late regions (e.g., the L1-L5 regions), genes involvedin viral packaging (e.g., the IVa2 gene), and virus-associated RNAs(e.g., VA-RNA1 and/or VA-RNA-2).

The replication-deficient adenoviral vector desirably requirescomplementation of at least one replication-essential gene function ofone or more regions of the adenoviral genome. Preferably, the adenoviralvector requires complementation of at least one gene function of the E1Aregion, the E1B region, or the E4 region of the adenoviral genomerequired for viral replication (denoted an E1-deficient or E4-deficientadenoviral vector). In addition to a deficiency in the E1 region, therecombinant adenovirus also can have a mutation in the major latepromoter (MLP), as discussed in International Patent ApplicationPublication WO 00/00628. Most preferably, the adenoviral vector isdeficient in at least one replication-essential gene function (desirablyall replication-essential gene functions) of the E1 region and at leastone gene function of the nonessential E3 region (e.g., an Xba I deletionof the E3 region) (denoted an E1/E3-deficient adenoviral vector). Withrespect to the E1 region, the adenoviral vector can be deficient in partor all of the E1A region and/or part or all of the E1B region, e.g., inat least one replication-essential gene function of each of the E1A andE1B regions, thus requiring complementation of the E1A region and theE1B region of the adenoviral genome for replication. The adenoviralvector also can require complementation of the E4 region of theadenoviral genome for replication, such as through a deficiency in oneor more replication-essential gene functions of the E4 region.

When the adenoviral vector is E1-deficient, the adenoviral vector genomecan comprise a deletion beginning at any nucleotide between nucleotides335 to 375 (e.g., nucleotide 356) and ending at any nucleotide betweennucleotides 3,310 to 3,350 (e.g., nucleotide 3,329) or even ending atany nucleotide between 3,490 and 3,530 (e.g., nucleotide 3,510) (basedon the adenovirus serotype 5 genome). When E2A-deficient, the adenoviralvector genome can comprise a deletion beginning at any nucleotidebetween nucleotides 22,425 to 22,465 (e.g., nucleotide 22,443) andending at any nucleotide between nucleotides 24,010 to 24,050 (e.g.,nucleotide 24,032) (based on the adenovirus serotype 5 genome). WhenE3-deficient, the adenoviral vector genome can comprise a deletionbeginning at any nucleotide between nucleotides 28,575 to 29,615 (e.g.,nucleotide 28,593) and ending at any nucleotide between nucleotides30,450 to 30,490 (e.g., nucleotide 30,470) (based on the adenovirusserotype 5 genome). When E4-deficient, the adenoviral vector genome cancomprise a deletion beginning at, for example, any nucleotide betweennucleotides 32,805 to 32,845 (e.g., nucleotide 32,826) and ending at,for example, any nucleotide between nucleotides 35,540 to 35,580 (e.g.,nucleotide 35,561) (based on the adenovirus serotype 5 genome). Theendpoints defining the deleted nucleotide portions can be difficult toprecisely determine and typically will not significantly affect thenature of the adenoviral vector, i.e., each of the aforementionednucleotide numbers can be ±1, 2, 3, 4, 5, or even 10 or 20 nucleotides.

When the adenoviral vector is deficient in at least onereplication-essential gene function in one region of the adenoviralgenome (e.g., an E1- or E1/E3-deficient adenoviral vector), theadenoviral vector is referred to as “singly replication-deficient.” Aparticularly preferred singly replication-deficient adenoviral vectoris, for example, a replication-deficient adenoviral vector requiring, atmost, complementation of the E1 region of the adenoviral genome, so asto propagate the adenoviral vector (e.g., to form adenoviral vectorparticles).

The adenoviral vector can be “multiply replication-deficient,” meaningthat the adenoviral vector is deficient in one or morereplication-essential gene functions in each of two or more regions ofthe adenoviral genome, and requires complementation of those functionsfor replication. For example, the aforementioned E1-deficient orE1/E3-deficient adenoviral vector can be further deficient in at leastone replication-essential gene function of the E4 region (denoted anE1/E4- or E1/E3/E4-deficient adenoviral vector), and/or the E2 region(denoted an E1/E2- or E1/E2/E3-deficient adenoviral vector), preferablythe E2A region (denoted an E1/E2A- or E1/E2A/E3-deficient adenoviralvector). When the adenoviral vector is multiply replication-deficient,the deficiencies can be a combination of the nucleotide deletionsdiscussed above with respect to each individual region. An adenoviralvector deleted of the entire E4 region can elicit a lower host immuneresponse.

If the adenoviral vector of the invention is deficient in areplication-essential gene function of the E2A region, the vectorpreferably does not comprise a complete deletion of the E2A region,which deletion preferably is less than about 230 base pairs in length.Generally, the E2A region of the adenovirus codes for a DBP (DNA bindingprotein), a polypeptide required for DNA replication. DBP is composed of473 to 529 amino acids depending on the viral serotype. It is believedthat DBP is an asymmetric protein that exists as a prolate ellipsoidconsisting of a globular Ct with an extended Nt domain. Studies indicatethat the Ct domain is responsible for DBP's ability to bind to nucleicacids, bind to zinc, and function in DNA synthesis at the level of DNAchain elongation. However, the Nt domain is believed to function in lategene expression at both transcriptional and post-transcriptional levels,is responsible for efficient nuclear localization of the protein, andalso may be involved in enhancement of its own expression. Deletions inthe Nt domain between amino acids 2 to 38 have indicated that thisregion is important for DBP function (Brough et al., Virology, 196:269-281 (1993)). While deletions in the E2A region coding for the Ctregion of the DBP have no effect on viral replication, deletions in theE2A region which code for amino acids 2 to 38 of the Nt domain of theDBP impair viral replication. It is preferable that any multiplyreplication-deficient adenoviral vector contains this portion of the E2Aregion of the adenoviral genome. In particular, for example, the desiredportion of the E2A region to be retained is that portion of the E2Aregion of the adenoviral genome which is defined by the 5′ end of theE2A region, specifically positions Ad5(23816) to Ad5(24032) of the E2Aregion of the adenoviral genome of serotype Ad5. This portion of theadenoviral genome desirably is included in the adenoviral vector becauseit is not complemented in current E2A cell lines so as to provide thedesired level of viral propagation.

While the above-described deletions are described with respect to anadenovirus serotype 5 genome, one of ordinary skill in the art candetermine the nucleotide coordinates of the same regions of otheradenovirus serotypes, such as an adenovirus serotype 2 genome, withoutundue experimentation, based on the similarity between the genomes ofvarious adenovirus serotypes, particularly adenovirus serotypes 2 and 5.

In one embodiment of the invention, the adenoviral vector can comprisean adenoviral genome deficient in one or more replication-essential genefunctions of each of the E1 and E4 regions (i.e., the adenoviral vectoris an E1/E4-deficient adenoviral vector), preferably with the entirecoding region of the E4 region having been deleted from the adenoviralgenome. In other words, all the open reading frames (ORFs) of the E4region have been removed. Most preferably, the adenoviral vector isrendered replication-deficient by deletion of all of the E1 region andby deletion of a portion of the E4 region. The E4 region of theadenoviral vector can retain the native E4 promoter, polyadenylationsequence, and/or the right-side inverted terminal repeat (ITR).

It should be appreciated that the deletion of different regions of theadenoviral vector can alter the immune response of the mammal. Inparticular, deletion of different regions can reduce the inflammatoryresponse generated by the adenoviral vector. Furthermore, the adenoviralvector's coat protein can be modified so as to decrease the adenoviralvector's ability or inability to be recognized by a neutralizingantibody directed against the wild-type coat protein, as described inInternational Patent Application WO 98/40509. Such modifications areuseful for long-term treatment of persistent disorders.

The adenoviral vector, when multiply replication-deficient, especiallyin replication-essential gene functions of the E1 and E4 regions, caninclude a spacer sequence to provide viral growth in a complementingcell line similar to that achieved by singly replication-deficientadenoviral vectors, particularly an E1-deficient adenoviral vector. In apreferred E4-deficient adenoviral vector of the invention wherein the L5fiber region is retained, the spacer is desirably located between the L5fiber region and the right-side ITR. More preferably in such anadenoviral vector, the E4 polyadenylation sequence alone or, mostpreferably, in combination with another sequence exists between the L5fiber region and the right-side ITR, so as to sufficiently separate theretained L5 fiber region from the right-side ITR, such that viralproduction of such a vector approaches that of a singlyreplication-deficient adenoviral vector, particularly a singlyreplication-deficient E1 deficient adenoviral vector.

The spacer sequence can contain any nucleotide sequence or sequenceswhich are of a desired length, such as sequences at least about 15 basepairs (e.g., between about 15 base pairs and about 12,000 base pairs),preferably about 100 base pairs to about 10,000 base pairs, morepreferably about 500 base pairs to about 8,000 base pairs, even morepreferably about 1,500 base pairs to about 6,000 base pairs, and mostpreferably about 2,000 to about 3,000 base pairs in length. The spacersequence can be coding or non-coding and native or non-native withrespect to the adenoviral genome, but does not restore thereplication-essential function to the deficient region. The spacer canalso contain a promoter-variable expression cassette. More preferably,the spacer comprises an additional polyadenylation sequence and/or apassenger gene. Preferably, in the case of a spacer inserted into aregion deficient for E4, both the E4 polyadenylation sequence and the E4promoter of the adenoviral genome or any other (cellular or viral)promoter remain in the vector. The spacer is located between the E4polyadenylation site and the E4 promoter, or, if the E4 promoter is notpresent in the vector, the spacer is proximal to the right-side ITR. Thespacer can comprise any suitable polyadenylation sequence. Examples ofsuitable polyadenylation sequences include synthetic optimizedsequences, BGH (Bovine Growth Hormone), polyoma virus, TK (ThymidineKinase), EBV (Epstein Barr Virus) and the papillomaviruses, includinghuman papillomaviruses and BPV (Bovine Papilloma Virus). Preferably,particularly in the E4 deficient region, the spacer includes an SV40polyadenylation sequence. The SV40 polyadenylation sequence allows forhigher virus production levels of multiply replication deficientadenoviral vectors. In the absence of a spacer, production of fiberprotein and/or viral growth of the multiply replication-deficientadenoviral vector is reduced by comparison to that of a singlyreplication-deficient adenoviral vector. However, inclusion of thespacer in at least one of the deficient adenoviral regions, preferablythe E4 region, can counteract this decrease in fiber protein productionand viral growth. Ideally, the spacer is composed of the glucuronidasegene. The use of a spacer in an adenoviral vector is further describedin, for example, U.S. Pat. No. 5,851,806 and International PatentApplication WO 97/21826.

It has been observed that an at least E4-deficient adenoviral vectorexpresses a transgene at high levels for a limited amount of time invivo and that persistence of expression of a transgene in an at leastE4-deficient adenoviral vector can be modulated through the action of atrans-acting factor, such as HSV ICP0, Ad pTP, CMV-IE2, CMV-IE86, HIVtat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205osteosarcoma cell line that functions like HSV ICP0, or the cellularfactor in PC12 cells that is induced by nerve growth factor, amongothers, as described in for example, U.S. Pat. Nos. 6,225,113,6,649,373, and 6,660,521, and International Patent ApplicationPublication WO 00/34496. In view of the above, a replication-deficientadenoviral vector (e.g., the at least E4-deficient adenoviral vector) ora second expression vector can comprise a nucleic acid sequence encodinga trans-acting factor that modulates the persistence of expression ofthe nucleic acid sequence. Persistent expression of antigenic DNA can bedesired when generating immune tolerance.

Desirably, the adenoviral vector requires, at most, complementation ofreplication-essential gene functions of the E1, E2A, and/or E4 regionsof the adenoviral genome for replication (i.e., propagation). However,the adenoviral genome can be modified to disrupt one or morereplication-essential gene functions as desired by the practitioner, solong as the adenoviral vector remains deficient and can be propagatedusing, for example, complementing cells and/or exogenous DNA (e.g.,helper adenovirus) encoding the disrupted replication-essential genefunctions. In this respect, the adenoviral vector can be deficient inreplication-essential gene functions of only the early regions of theadenoviral genome, only the late regions of the adenoviral genome, andboth the early and late regions of the adenoviral genome. Suitablereplication-deficient adenoviral vectors, including singly and multiplyreplication-deficient adenoviral vectors, are disclosed in U.S. Pat.Nos. 5,837,511, 5,851,806, 5,994,106, 6,127,175, and 6,482,616; U.S.Patent Application Publications 2001/0043922 A1, 2002/0004040 A1,2002/0031831 A1, 2002/0110545 A1, and 2004/0161848 A1; and InternationalPatent Application Publications WO 94/28152, WO 95/02697, WO 95/16772,WO 95/34671, WO 96/22378, WO 97/12986, WO 97/21826, and WO 03/022311.

By removing all or part of, for example, the E1, E3, and E4 regions ofthe adenoviral genome, the resulting adenoviral vector is able to acceptinserts of exogenous nucleic acid sequences while retaining the abilityto be packaged into adenoviral capsids. The nucleic acid sequence can bepositioned in the E1 region, the E3 region, or the E4 region of theadenoviral genome. Indeed, the nucleic acid sequence can be insertedanywhere in the adenoviral genome so long as the position does notprevent expression of the nucleic acid sequence or interfere withpackaging of the adenoviral vector.

Replication-deficient adenoviral vectors are typically produced incomplementing cell lines that provide gene functions not present in thereplication-deficient adenoviral vectors, but required for viralpropagation, at appropriate levels in order to generate high titers ofviral vector stock. Desirably, the complementing cell line comprises,integrated into the cellular genome, adenoviral nucleic acid sequenceswhich encode gene functions required for adenoviral propagation. Apreferred cell line complements for at least one and preferably allreplication-essential gene functions not present in areplication-deficient adenovirus. The complementing cell line cancomplement for a deficiency in at least one replication-essential genefunction encoded by the early regions, late regions, viral packagingregions, virus-associated RNA regions, or combinations thereof,including all adenoviral functions (e.g., to enable propagation ofadenoviral amplicons). Most preferably, the complementing cell linecomplements for a deficiency in at least one replication-essential genefunction (e.g., two or more replication-essential gene functions) of theE1 region of the adenoviral genome, particularly a deficiency in areplication-essential gene function of each of the E1A and E1B regions.In addition, the complementing cell line can complement for a deficiencyin at least one replication-essential gene function of the E2(particularly as concerns the adenoviral DNA polymerase and terminalprotein) and/or E4 regions of the adenoviral genome. Desirably, a cellthat complements for a deficiency in the E4 region comprises the E4-ORF6gene sequence and produces the E4-ORF6 protein. Such a cell desirablycomprises at least ORF6 and no other ORF of the E4 region of theadenoviral genome. The cell line preferably is further characterized inthat it contains the complementing genes in a non-overlapping fashionwith the adenoviral vector, which minimizes, and practically eliminates,the possibility of the vector genome recombining with the cellular DNA.Accordingly, the presence of replication competent adenoviruses (RCA) isminimized if not avoided in the vector stock, which, therefore, issuitable for certain therapeutic purposes, especially vaccinationpurposes. The lack of RCA in the vector stock avoids the replication ofthe adenoviral vector in non-complementing cells. Construction of such acomplementing cell lines involve standard molecular biology and cellculture techniques, such as those described by Sambrook et al.,Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994).

Complementing cell lines for producing the adenoviral vector include,but are not limited to, 293 cells (described in, e.g., Graham et al., J.Gen. Virol., 36: 59-72 (1977)), PER.C6 cells (described in, e.g.,International Patent Application Publication WO 97/00326, and U.S. Pat.Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g.,International Patent Application Publication WO 95/34671 and Brough etal., J. Virol., 71: 9206-9213 (1997)). Additional complementing cellsare described in, for example, U.S. Pat. Nos. 6,677,156 and 6,682,929,and International Patent Application Publication WO 03/20879. In someinstances, the cellular genome need not comprise nucleic acid sequences,the gene products of which complement for all of the deficiencies of areplication-deficient adenoviral vector. One or morereplication-essential gene functions lacking in a replication-deficientadenoviral vector can be supplied by a helper virus, e.g., an adenoviralvector that supplies in trans one or more essential gene functionsrequired for replication of the desired adenoviral vector. Helper virusis often engineered to prevent packaging of infectious helper virus. Forexample, one or more replication-essential gene functions of the E1region of the adenoviral genome are provided by the complementing cell,while one or more replication-essential gene functions of the E4 regionof the adenoviral genome are provided by a helper virus.

If the adenoviral vector is not replication-deficient, ideally theadenoviral vector is manipulated to limit replication of the vector towithin a target tissue. The adenoviral vector can be aconditionally-replicating adenoviral vector, which is engineered toreplicate under conditions pre-determined by the practitioner. Forexample, replication-essential gene functions, e.g., gene functionsencoded by the adenoviral early regions, can be operably linked to aninducible, repressible, or tissue-specific transcription controlsequence, e.g., promoter. In this embodiment, replication requires thepresence or absence of specific factors that interact with thetranscription control sequence. In autoimmune disease treatment, it canbe advantageous to control adenoviral vector replication in, forinstance, lymph nodes, to obtain continual antigen production andcontrol immune cell production. Conditionally-replicating adenoviralvectors are described further in U.S. Pat. No. 5,998,205.

In addition to modification (e.g., deletion, mutation, or replacement)of adenoviral sequences encoding replication-essential gene functions,the adenoviral genome can contain benign or non-lethal modifications,i.e., modifications which do not render the adenovirusreplication-deficient, or, desirably, do not adversely affect viralfunctioning and/or production of viral proteins, even if suchmodifications are in regions of the adenoviral genome that otherwisecontain replication-essential gene functions. Such modificationscommonly result from DNA manipulation or serve to facilitate expressionvector construction. For example, it can be advantageous to remove orintroduce restriction enzyme sites in the adenoviral genome. Such benignmutations often have no detectable adverse effect on viral functioning.For example, the adenoviral vector can comprise a deletion ofnucleotides 10,594 and 10,595 (based on the adenoviral serotype 5genome), which are associated with VA-RNA-1 transcription, but thedeletion of which does not prohibit production of VA-RNA-1.

Similarly, it will be appreciated that numerous adenoviral vectors areavailable commercially. Construction of adenoviral vectors is wellunderstood in the art. Adenoviral vectors can be constructed and/orpurified using methods known in the art (e.g., using complementing celllines, such as the 293 cell line, PER.C6 cell line, or 293-ORF6 cellline) and methods set forth, for example, in U.S. Pat. Nos. 5,965,358,5,994,128, 6,033,908, 6,168,941, 6,329,200, 6,383,795, 6,440,728,6,447,995, 6,475,757, 6,586,226, 6,908,762, and 6,913,927; andInternational Patent Applications WO 98/53087, WO 98/56937, WO 99/15686,WO 99/54441, WO 00/12765, WO 01/77304, and WO 02/29388, as well as theother references identified herein.

In another embodiment, the coat protein of a viral vector, preferably anadenoviral vector, can be manipulated to alter the binding specificityor recognition of a virus for a viral receptor on a potential host cell.For adenovirus, such manipulations can include deletion of regions ofthe fiber, penton, or hexon, insertions of various native or non-nativeligands into portions of the coat protein, and the like. Manipulation ofthe coat protein can broaden the range of cells infected by a viralvector or enable targeting of a viral vector to a specific cell type.

Any suitable technique for altering native binding to a host cell, suchas native binding of the fiber protein to the coxsackievirus andadenovirus receptor (CAR) of a cell, can be employed. For example,differing fiber lengths can be exploited to ablate native binding tocells. This optionally can be accomplished via the addition of a bindingsequence to the penton base or fiber knob. This addition of a bindingsequence can be done either directly or indirectly via a bispecific ormultispecific binding sequence. In an alternative embodiment, theadenoviral fiber protein can be modified to reduce the number of aminoacids in the fiber shaft, thereby creating a “short-shafted” fiber (asdescribed in, for example, U.S. Pat. No. 5,962,311). Use of anadenovirus comprising a short-shafted adenoviral fiber gene reduces thelevel or efficiency of adenoviral fiber binding to its cell-surfacereceptor and increases adenoviral penton base binding to itscell-surface receptor, thereby increasing the specificity of binding ofthe adenovirus to a given cell. Alternatively, use of an adenoviruscomprising a short-shafted fiber enables targeting of the adenovirus toa desired cell-surface receptor by the introduction of a non-nativeamino acid sequence either into the penton base or the fiber knob.

In another embodiment, the nucleic acid residues encoding amino acidresidues associated with native substrate binding can be changed,supplemented, or deleted (see, e.g., International Patent ApplicationPublication WO 00/15823, Einfeld et al., J. Virol., 75(23): 11284-11291(2001), and van Beusechem et al., J. Virol., 76(6): 2753-2762 (2002))such that the adenoviral vector incorporating the mutated nucleic acidresidues (or having the fiber protein encoded thereby) is less able tobind its native substrate. In this respect, the native CAR and integrinbinding sites of the adenoviral vector, such as the knob domain of theadenoviral fiber protein and an Arg-Gly-Asp (RGD) sequence located inthe adenoviral penton base, respectively, can be removed or disrupted.Any suitable amino acid residue(s) of a fiber protein that mediates orassists in the interaction between the knob and CAR can be mutated orremoved, so long as the fiber protein is able to trimerize. Similarly,amino acids can be added to the fiber knob as long as the fiber proteinretains the ability to trimerize. Suitable residues include amino acidswithin the exposed loops of the serotype 5 fiber knob domain, such as,for example, the AB loop, the DE loop, the FG loop, and the HI loop,which are further described in, for example, Roelvink et al., Science,286: 1568-1571 (1999), and U.S. Pat. No. 6,455,314. Any suitable aminoacid residue(s) of a penton base protein that mediates or assists in theinteraction between the penton base and integrins can be mutated orremoved. Suitable residues include, for example, one or more of the fiveRGD amino acid sequence motifs located in the hypervariable region ofthe Ad5 penton base protein (as described, for example, U.S. Pat. No.5,731,190). The native integrin binding sites on the penton base proteinalso can be disrupted by modifying the nucleic acid sequence encodingthe native RGD motif such that the native RGD amino acid sequence isconformationally inaccessible for binding to the αv integrin receptor,such as by inserting a DNA sequence into or adjacent to the nucleic acidsequence encoding the adenoviral penton base protein. Preferably, theadenoviral vector comprises a fiber protein and a penton base proteinthat do not bind to CAR and integrins, respectively. Alternatively, theadenoviral vector comprises fiber protein and a penton base protein thatbind to CAR and integrins, respectively, but with less affinity than thecorresponding wild type coat proteins. The adenoviral vector exhibitsreduced binding to CAR and integrins if a modified adenoviral fiberprotein and penton base protein binds CAR and integrins, respectively,with at least about 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, or100-fold less affinity than a non-modified adenoviral fiber protein andpenton base protein of the same serotype.

The adenoviral vector also can comprise a chimeric coat proteincomprising a non-native amino acid sequence that binds a substrate(i.e., a ligand), such as a cellular receptor other than CAR the αvintegrin receptor. Such a chimeric coat protein allows an adenoviralvector to bind, and desirably, infect host cells not naturally infectedby the corresponding adenovirus that retains the ability to bind nativecell surface receptors, thereby further expanding the repertoire of celltypes infected by the adenoviral vector. The non-native amino acidsequence of the chimeric adenoviral coat protein allows an adenoviralvector comprising the chimeric coat protein to bind and, desirably,infect host cells not naturally infected by a corresponding adenoviruswithout the non-native amino acid sequence (i.e., host cells notinfected by the corresponding wild-type adenovirus), to bind to hostcells naturally infected by the corresponding adenovirus with greateraffinity than the corresponding adenovirus without the non-native aminoacid sequence, or to bind to particular target cells with greateraffinity than non-target cells. A “non-native” amino acid sequence cancomprise an amino acid sequence not naturally present in the adenoviralcoat protein or an amino acid sequence found in the adenoviral coat butlocated in a non-native position within the capsid. By “preferentiallybinds” is meant that the non-native amino acid sequence binds areceptor, such as, for instance, αvβ3 integrin, with at least about3-fold greater affinity (e.g., at least about 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 35-fold, 45-fold, or 50-fold greater affinity) thanthe non-native ligand binds a different receptor, such as, for instance,αvβ1 integrin.

Desirably, the adenoviral vector comprises a chimeric coat proteincomprising a non-native amino acid sequence that confers to the chimericcoat protein the ability to bind to an immune cell more efficiently thana wild-type adenoviral coat protein. In particular, the adenoviralvector can comprise a chimeric adenoviral fiber protein comprising anon-native amino acid sequence which facilitates uptake of theadenoviral vector by immune cells, preferably antigen presenting cells,such as dendritic cells, monocytes, and macrophages. In a preferredembodiment, the adenoviral vector comprises a chimeric fiber proteincomprising an amino acid sequence (e.g., a non-native amino acidsequence) comprising an RGD motif including, but not limited to, CRGDC(SEQ ID NO: 1), CXCRGDCXC (SEQ ID NO: 2), wherein X represents any aminoacid, and CDCRGDCFC (SEQ ID NO: 3), which increases transductionefficiency of an adenoviral vector into dendritic cells. The RGD-motif,or any non-native amino acid sequence, preferably is inserted into theadenoviral fiber knob region, ideally in an exposed loop of theadenoviral knob, such as the HI loop. A non-native amino acid sequencealso can be appended to the C-terminus of the adenoviral fiber protein,optionally via a spacer sequence. The spacer sequence preferablycomprises between one and two-hundred amino acids, and can (but neednot) have an intended function.

Where dendritic cells are the desired target cell, the non-native aminoacid sequence can optionally recognize a protein typically found ondendritic cell surfaces such as adhesion proteins, chemokine receptors,complement receptors, co-stimulation proteins, cytokine receptors, highlevel antigen presenting molecules, homing proteins, marker proteins,receptors for antigen uptake, signaling proteins, virus receptors, etc.Examples of such potential ligand-binding sites in dendritic cellsinclude αvβ3 integrins, αvβ5 integrins, 2A1, 7-TM receptors, CD1, CD11a,CD11b, CD11c, CD21, CD24, CD32, CD4, CD40, CD44 variants, CD46, CD49d,CD50, CD54, CD58, CD64, ASGPR, CD80, CD83, CD86, E-cadherin, integrins,M342, MHC-I, MHC-II, MIDC-8, MMR, OX62, p200-MR6, p55, S100, TNF-R, etc.Where dendritic cells are targeted, the ligand preferably recognizes theCD40 cell surface protein, such as, for example, by way of a CD-40(bi)specific antibody fragment or by way of a domain derived from theCD40L polypeptide.

Where macrophages are the desired target, the non-native amino acidsequence optionally can recognize a protein typically found onmacrophage cell surfaces, such as phosphatidylserine receptors,vitronectin receptors, integrins, adhesion receptors, receptors involvedin signal transduction and/or inflammation, markers, receptors forinduction of cytokines, or receptors up-regulated upon challenge bypathogens, members of the group B scavenger receptor cysteine-rich(SRCR) superfamily, sialic acid binding receptors, members of the Fcreceptor family, B7-1 and B7-2 surface molecules, lymphocyte receptors,leukocyte receptors, antigen presenting molecules, and the like.Examples of suitable macrophage surface target proteins include, but arenot limited to, heparin sulfate proteoglycans, αvβ3 integrins, αvβ5integrins, B7-1, B7-2, CD11c, CD13, CD16, CD163, CD1a, CD22, CD23, CD29,Cd32, CD33, CD36, CD44, CD45, CD49e, CD52, CD53, CD54, CD71, CD87, CD9,CD98, Ig receptors, Fc receptor proteins (e.g., subtypes of Fcα, Fcγ,Fcε, etc.), folate receptor b, HLA Class I, Sialoadhesin, siglec-5, andthe toll-like receptor-2 (TLR2).

Where B-cells are the desired target, the non-native amino acid sequencecan recognize a protein typically found on B-cell surfaces, such asintegrins and other adhesion molecules, complement receptors,interleukin receptors, phagocyte receptors, immunoglobulin receptors,activation markers, transferrin receptors, members of the scavengerreceptor cysteine-rich (SRCR) superfamily, growth factor receptors,selectins, MHC molecules, TNF-receptors, and TNF-R associated factors.Examples of typical B-cell surface proteins include β-glycan, B cellantigen receptor (BAC), B7-2, B-cell receptor (BCR), C3d receptor, CD1,CD18, CD19, CD20, CD21, CD22, CD23, CD35, CD40, CD5, CD6, CD69, CD69,CD71, CD79a/CD79b dimer, CD95, endoglin, Fas antigen, human Igreceptors, Fc receptor proteins (e.g., subtypes of Fcα, Fcγ, Fcε, etc.),IgM, gp200-MR6, Growth Hormone Receptor (GHi-R), ICAM-1, ILT2, CD85, MHCclass I and II molecules, transforming growth factor receptor (TGF-R),α4β7 integrin, and αvβ3 integrin.

In another embodiment of the invention, the adenoviral vector comprisesa chimeric virus coat protein not selective for a specific type ofeukaryotic cell. The chimeric coat protein differs from the wild-typecoat protein by an insertion of a non-native amino acid sequence into orin place of an internal coat protein sequence. In this embodiment, thechimeric adenovirus coat protein efficiently binds to a broader range ofeukaryotic cells than a wild-type adenovirus coat, such as described inInternational Patent Application WO 97/20051.

The ability of an adenoviral vector to recognize a potential host cellcan be modulated without genetic manipulation of the coat protein, i.e.,through use of a bi-specific molecule. For instance, complexing anadenovirus with a bispecific molecule comprising a penton base-bindingdomain and a domain that selectively binds a particular cell surfacebinding site enables the targeting of the adenoviral vector to aparticular cell type. Likewise, an antigen can be conjugated to thesurface of the adenoviral particle through non-genetic means.

A non-native amino acid sequence can be conjugated to any of theadenoviral coat proteins to form a chimeric adenoviral coat protein.Therefore, for example, a non-native amino acid sequence can beconjugated to, inserted into, or attached to a fiber protein, a pentonbase protein, a hexon protein, proteins IX, VI, or IIIa, etc. Thesequences of such proteins, and methods for employing them inrecombinant proteins, are well known in the art (see, e.g., U.S. Pat.Nos. 5,543,328; 5,559,099; 5,712,136; 5,731,190; 5,756,086; 5,770,442;5,846,782; 5,962,311; 5,965,541; 5,846,782; 6,057,155; 6,127,525;6,153,435; 6,329,190; 6,455,314; 6,465,253; 6,576,456; 6,649,407;6,740,525, and International Patent Application Publications WO96/07734, WO 96/26281, WO 97/20051, WO 98/07877, WO 98/07865, WO98/40509, WO 98/54346, WO 00/15823, WO 01/58940, and WO 01/92549). Thechimeric adenoviral coat protein can be generated using standardrecombinant DNA techniques known in the art. Preferably, the nucleicacid sequence encoding the chimeric adenoviral coat protein is locatedwithin the adenoviral genome and is operably linked to a promoter thatregulates expression of the coat protein in a wild-type adenovirus.Alternatively, the nucleic acid sequence encoding the chimericadenoviral coat protein is located within the adenoviral genome and ispart of an expression cassette which comprises genetic elements requiredfor efficient expression of the chimeric coat protein.

The coat protein portion of the chimeric adenovirus coat protein can bea full-length adenoviral coat protein to which the ligand domain isappended, or it can be truncated, e.g., internally or at the C- and/orN-terminus. However modified (including the presence of the non-nativeamino acid), the chimeric coat protein preferably is able to incorporateinto an adenoviral capsid. Where the non-native amino acid sequence isattached to the fiber protein, preferably it does not disturb theinteraction between viral proteins or fiber monomers. Thus, thenon-native amino acid sequence preferably is not itself anoligomerization domain, as such can adversely interact with thetrimerization domain of the adenovirus fiber. Preferably the non-nativeamino acid sequence is added to the virion protein, and is incorporatedin such a manner as to be readily exposed to a substrate, cellsurface-receptor, or immune cell (e.g., at the N- or C-terminus of theadenoviral protein, attached to a residue facing a substrate, positionedon a peptide spacer, etc.) to maximally expose the non-native amino acidsequence. Ideally, the non-native amino acid sequence is incorporatedinto an adenoviral fiber protein at the C-terminus of the fiber protein(and attached via a spacer) or incorporated into an exposed loop (e.g.,the HI loop) of the fiber to create a chimeric coat protein. Where thenon-native amino acid sequence is attached to or replaces a portion ofthe penton base, preferably it is within the hypervariable regions toensure that it contacts the substrate, cell surface receptor, or immunecell. Where the non-native amino acid sequence is attached to the hexon,preferably it is within a hypervariable region (Crawford-Miksza et al.,J. Virol., 70 (3): 1836-44 (1996)). Where the non-native amino acid isattached to or replaces a portion of pIX, preferably it is within theC-terminus of pIX. Use of a spacer sequence to extend the non-nativeamino acid sequence away from the surface of the adenoviral particle canbe advantageous in that the non-native amino acid sequence can be moreavailable for binding to a receptor, and any steric interactions betweenthe non-native amino acid sequence and the adenoviral fiber monomers canbe reduced.

Binding affinity of a non-native amino acid sequence to a cellularreceptor can be determined by any suitable assay, a variety of whichassays are known and are useful in selecting a non-native amino acidsequence for incorporating into an adenoviral coat protein. Desirably,the transduction levels of host cells are utilized in determiningrelative binding efficiency. Thus, for example, host cells displayingαvβ3 integrin on the cell surface (e.g., MDAMB435 cells) can be exposedto an adenoviral vector comprising the chimeric coat protein and thecorresponding adenovirus without the non-native amino acid sequence, andthen transduction efficiencies can be compared to determine relativebinding affinity. Similarly, both host cells displaying αvβ3 integrin onthe cell surface (e.g., MDAMB435 cells) and host cells displayingpredominantly αvβ1 on the cell surface (e.g., 293 cells) can be exposedto the adenoviral vectors comprising the chimeric coat protein, and thentransduction efficiencies can be compared to determine binding affinity.

In other embodiments (e.g., to facilitate purification or propagationwithin a specific engineered cell type), a non-native amino acid (e.g.,ligand) can bind a compound other than a cell-surface protein. Thus, theligand can bind blood- and/or lymph-borne proteins (e.g., albumin),synthetic peptide sequences such as polyamino acids (e.g., polylysine,polyhistidine, etc.), artificial peptide sequences (e.g., FLAG), and RGDpeptide fragments (Pasqualini et al., J. Cell. Biol, 130: 1189 (1995)).A ligand can even bind non-peptide substrates, such as plastic (e.g.,Adey et al., Gene, 156: 27 (1995)), biotin (Saggio et al., Biochem. J.,293: 613 (1993)), a DNA sequence (Cheng et al., Gene, 171: 1 (1996), andKrook et al., Biochem. Biophys., Res. Commun., 204: 849 (1994)),streptavidin (Geibel et al., Biochemistry, 34: 15430 (1995), and Katz,Biochemistry, 34: 15421 (1995)), nitrostreptavidin (Balass et al., Anal.Biochem., 243: 264 (1996)), heparin (Wickham et al., Nature Biotechnol.,14: 1570-73 (1996)), and other substrates.

Disruption of native binding of adenoviral coat proteins to a cellsurface receptor can also render it less able to interact with theinnate or acquired host immune system. Aside from pre-existing immunity,adenoviral vector administration induces inflammation and activates bothinnate and acquired immune mechanisms. Adenoviral vectors activateantigen-specific (e.g., T-cell dependent) immune responses, which limitthe duration of transgene expression following an initial administrationof the vector. In addition, exposure to adenoviral vectors stimulatesproduction of neutralizing antibodies by B cells, which can precludegene expression from subsequent doses of adenoviral vector (Wilson andKay, Nat. Med., 3(9): 887-889 (1995)). Indeed, the effectiveness ofrepeated administration of the vector can be severely limited by hostimmunity. In addition to stimulation of humoral immunity, cell-mediatedimmune functions are responsible for clearance of the virus from thebody. Rapid clearance of the virus is attributed to innate immunemechanisms (see, e.g., Worgall et al., Human Gene Therapy, 8: 37-44(1997)), and likely involves Kupffer cells found within the liver. Thus,by ablating native binding of an adenovirus fiber protein and pentonbase protein, immune system recognition of an adenoviral vector isdiminished, thereby increasing vector tolerance by the host.

Suitable modifications to an adenoviral vector are described in U.S.Pat. Nos. 5,543,328, 5,559,099, 5,712,136, 5,731,190, 5,756,086,5,770,442, 5,846,782, 5,871,727, 5,885,808, 5,922,315, 5,962,311,5,965,541, 6,057,155, 6,127,525, 6,153,435, 6,329,190, 6,455,314,6,465,253, 6,576,456, 6,596,270, 6,649,407, 6,740,525; 6,951,755; U.S.Patent Application Publications 2003/0099619 A1, 2003/0166286 A1, and2004/0161848 A1; and International Patent Applications WO 95/02697, WO95/16772, WO 95/34671, WO 96/07734, WO 96/22378, WO 96/26281, WO97/20051, WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, WO00/15823, WO 01/58940, and WO 01/92549.

Any type of nucleic acid sequence (e.g., DNA, RNA, and cDNA) that can beinserted into an adenoviral vector can be used in connection with theinvention. Preferably, each nucleic acid sequence is DNA, and preferablyencodes a protein (i.e., one or more nucleic acid sequences encoding oneor more proteins). In a particularly preferred embodiment, at least onenucleic acid sequence encodes an antigen. An “antigen” is a moleculethat induces an immune response in a mammal. An “immune response” canentail, for example, antibody production and/or the activation of immuneeffector cells (e.g., T cells). An antigen in the context of theinvention can comprise any subunit, fragment, or epitope of anyproteinaceous molecule, including a protein or peptide of viral,bacterial, parasitic, fungal, protozoan, prion, cellular, orextracellular origin, which ideally provokes an immune response inmammal, preferably leading to protective immunity. By “epitope” is meanta sequence on an antigen that is recognized by an antibody or an antigenreceptor. Epitopes also are referred to in the art as “antigenicdeterminants.”

In one embodiment, the antigen can be a viral antigen. The viral antigencan be isolated from any virus including, but not limited to, a virusfrom any of the following viral families: Arenaviridae, Arterivirus,Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae,Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus,Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae(e.g., Coronavirus, such as severe acute respiratory syndrome (SARS)virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus,Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g.,Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g.,Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, andDengue virus 4), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae(e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus),Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae,Orthomyxoviridae (e.g., Influenzavirus A and B), Papovaviridae,Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytialvirus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus,hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia virus),Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such ashuman immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae, andTotiviridae. Particularly preferred retroviridae (retrovirus) antigensinclude, for example, HIV antigens, such as all or part of the gag, env,or pol proteins, or fusion proteins comprising all or part of the gag,env, or pol proteins. Any clade of HIV is appropriate for antigenselection, including clades A, B, C, MN, and the like. Particularlypreferred coronavirus antigens include, for example, SARS virusantigens. Suitable SARS virus antigens for the invention include, forexample, all or part of the E protein, the M protein, and the spikeprotein of the SARS virus. Suitable viral antigens also include all orpart of Dengue protein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2,and Dengue D1NS3. The antigenic peptides specifically recited herein aremerely exemplary as any viral protein can be used in the context of theinvention.

In a preferred embodiment of the invention, the antigen is anaphthovirus antigen. Preferably, the antigen is a foot-and-mouth diseasevirus (FMDV) antigen. There are seven known serotypes of FMDV, and over60 known subtypes (see, e.g., Mahy, Curr. Top. Microbiol. Immunol., 288:1-8 (2005), and Musser, J. Am. Vet. Med. Assoc., 224(8): 1261-8 (2004)).FMDV antigens are generally known to those of skill in the art, andinclude, but are not limited to, antigens of FMDV serotypes A (e.g.,subtypes A24 and A12), O (e.g., subtypes O1C and O1M), C, Asia 1, SAT 1,SAT 2, and SAT 3. In a preferred embodiment, the antigen is obtainedfrom strain A24 Cruzeiro, Asia 1, O1C, or O1M. One of ordinary skill inthe art will appreciate that each FMDV serotype is antigenicallydistinct from the other serotypes. Furthermore, within each serotypethere is considerable antigenic diversity. Thus, antisera raised againstone strain of FMDV serotype may not recognize other strains of the sameserotype.

The FMDV particle consists of a single strand of RNA and fourpolypeptides, namely 1A, 1B, 1C, and 1D, collectively referred to as P1,which form the capsid proteins of the virus. The P1 protein also isreferred to in the art as VP 1, VPThr, and VPT (see Bachrach, et al., J.Immunology, 115: 1636-1641 (1975)), Strohmaier et al., Biochem. Biophys.Res. Comm., 85: 1640-1645 (1978), and Bachrach et al., Intervirology,12: 65-72 (1979)). It is generally considered that there areapproximately 60 copies of each capsid protein in the virus. Capsidprotein 1A is susceptible to cleavage when intact virus is treated withtrypsin, resulting in a large decrease in infectivity of most strains ofFMDV (see e.g., Wild et al., J. Gen. Virology, 1: 247-250 (1967)).Trypsin treatment may also reduce the capacity of virus to stimulate theproduction of neutralizing antibody. Thus, protein 1A likely is the mostimmunogenic structural FMDV protein, and is capable of elicitingeffective protection against infection by FMDV. In this regard, protein1A separated from virus particles has been shown to produce neutralizingantibodies and elicit effective protection against the virus (seeLaporte et al., C.R. Acad. Sc. Paris, 276: 3399-5401 (1973), andBachrach et al., Immunology, 115: 1636-1641 (1975)). The antigen alsocan be derived from a nonstructural protein of FMDV. FMDV nonstructuralproteins include the P2 protein (i.e., proteins 2A, 2B, and 2C) and theP3 protein (i.e., proteins 3A, 3B, 3C, and 3D). Antigenic peptides ofFMDV are disclosed in, for example, European Patent No. 0105481.

In a preferred embodiment of the invention, the antigen is an emptyvirus capsid of FMDV. An “empty virus capsid” contains only the portionof the FMDV genome encoding the viral structural proteins and the 3Cprotein, which is required for capsid formation (see Mayr et al.,Virology, 263: 496-506 (1999)), and does not contain the infectiousviral nucleic acid. Thus, animals inoculated with an empty virus capsidcan be distinguished from infected or convalescent animals usingapproved diagnostic assays (see, e.g., Mayr et al., Virology, 263:496-506 (1999), and Mayr et al., Vaccine, 19: 2152-2162 (2001)), as wellas with diagnostic assays using the most immunogenic non-structuralprotein, 3D (see, e.g., Pacheco et al., supra). Vaccination of swine andcattle with an empty viral capsid from FMDV strain A24 Cruzeirodelivered by an E1-deficient adenoviral vector can protect animals whenchallenged by direct inoculation of the heel bulb with virulenthomologous virus (see Moraes et al., Vaccine, 20: 1631-1639 (2002), andPacheco et al., supra).

When the antigen is an empty virus capsid of FMDV, the viral structuralproteins and the 3C protein preferably are derived from a virus of thesame serotype and subtype. For example, when the antigen is an emptyvirus capsid of the A24 Cruzeiro FMDV strain, both the virus structuralproteins and the 3C protein preferably are also of the A24 Cruzeiro FMDVstrain. Similarly, when the antigen is an empty virus capsid of theAsia1 serotype, both the virus structural proteins and the 3C proteinpreferably are also of the Asia1 serotype. When the antigen is an emptyvirus capsid of the O1C strain, both the virus structural proteins andthe 3C protein are also of the O1C strain. Antigens comprising FMDVempty virus capsids from different FMDV serotypes are also within thescope of the invention. In this respect, an FMDV empty virus capsid cancontain one or more virus structural proteins and/or a 3C proteinderived from a first FMDV serotype, and one or more virus structuralproteins and/or a 3C protein derived from a second FMDV serotype. Forexample, the antigen can comprise virus structural proteins from the A24Cruzeiro FMDV strain, while the 3C protein can be from the Asia1 strain.Similarly, the antigen can comprise virus structural proteins from theO1C strain, while the 3C protein can be from the A24 Cruzeiro strain.These specific embodiments, however, are merely exemplary. One ofordinary skill in the art will appreciate that genes from anycombination of FMDV serotypes can be utilized to generate the emptyvirus capsid antigen.

In addition to being an antigen itself, the FMDV empty capsid also canbe used in the invention as a virus-like particle (VLP) to deliver anantigen (e.g., a Plasmodium antigen, an HIV antigen, or a tumor antigen)to an appropriate host. A “virus-like particle” consists of one or moreviral coat proteins that assemble into viral particles, but lacks anyviral genetic material (see, e.g., Miyanohara et al., J. Virol., 59:176-180 (1986), Gheysen et al., Cell, 59: 103-112 (1989), and Buonaguroet al., ASHI Quarterly, 29: 78-80 (2005)). VLPs can be presented byantigen presenting cells (APCs) on MHC class II molecules, whichcorrelates with activation of CD4+ helper T cells. Recent evidence alsoindicates that VLPs can be presented on MHC class I molecules, therebyinducing CD8+ cytotoxic T cell activation (see, e.g., Moron et al., J.Immunol., 171: 2242-2250 (2003)). Thus, VLPs can elicit effective B celland T cell immune responses. In the context of the invention, theadenovirus comprises at least one nucleic acid sequence encoding an FMDVempty capsid, and at least one nucleic acid sequence encoding anantigen, wherein the nucleic acid sequence encoding the FMDV emptycapsid is modified so as to display the antigen on its surface. Thenucleic acid sequence encoding the FMDV empty capsid can modified in anysuitable manner. Preferably, the nucleic acid sequence encoding the FMDVempty capsid is modified using methods known in the art for altering thetropism of viral coat proteins, such as adenoviral coat proteins (e.g.,hexon protein).

When the empty capsid is used as a VLP, the empty capsid can deliver anysuitable antigen to a mammalian host. For example, the antigen can be atumor antigen. By “tumor antigen” is meant an antigen that is expressedby tumor cells but not normal cells, or an antigen that is expressed innormal cells but is overexpressed in tumor cells. Examples of suitabletumor antigens include, but are not limited to, β-catenin, BCR-ABLfusion protein, K-ras, N-ras, PTPRK, NY-ESO-1/LAGE-2, SSX-2, TRP2-INT2,CEA, gp100, kallikrein 4, prostate specific antigen (PSA), TRP-1/gp75,TRP-2, tyrosinase, EphA3, HER-2/neu, MUC1, p53, mdm-2, PSMA, RAGE-1,surviving, telomerase, and WT1. Other tumor antigens are known in theart and are described in, for example, The Peptide Database of T-CellDefined Tumor Antigens, maintained by the Ludwig Institute for CancerResearch (www.cancerimmunity.org/statics/databases.htm), Van den Eyndeet al., Curr. Opin. Immunol., 9: 684-93 (1997), Houghton et al., Curr.Opin. Immunol., 13: 134-140 (2001), and van der Bruggen et al., Immunol.Rev., 188: 51-64 (2002).

Alternatively, the empty capsid can be used to deliver a bacterialantigen to a mammalian host. The bacterial antigen can originate fromany bacterium including, but not limited to, Actinomyces, Anabaena,Bacillus, Bacteroides, Bdellovibrio, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Cytophaga, Deinococcus, Escherichia,Halobacterium, Heliobacter, Hyphomicrobium, Methanobacterium,Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, and Treponema.

The empty capsid also can be used to deliver a parasite antigen to amammalian host. The parasite antigen can originate from, for example, aparasite of the phylum Sporozoa (also referred to as phylumApicomplexa), Ciliophora, Rhizopoda, or Zoomastigophora. Preferably, theantigen is a parasite of the phylum Sporozoa and genus Plasmodium. Theantigen can be from any suitable Plasmodium species, but preferably isfrom a Plasmodium species that infects humans and causes malaria.Particularly preferred Plasmodium antigens include, for example,circumsporozoite protein (CSP), sporozoite surface protein 2 (SSP2),liver-stage antigen 1 (LSA-1), Pf exported protein 1 (PfExp-1)/Pyhepatocyte erythrocyte protein 17 (PyHEP 17), Pf Antigen 2, merozoitesurface protein 1 (MSP-1), merozoite surface protein 2 (MSP-2),erythrocyte binding antigen 175 (EBA-175), ring-infected erythrocytesurface antigen (RESA), serine repeat antigen (SERA), glycophorinbinding protein (GBP-130), histidine rich protein 2 (HRP-2),rhoptry-associated proteins 1 and 2 (RAP-1 and RAP-2), erythrocytemembrane protein 1 (PfEMP1), and apical membrane antigen 1 (AMA-1).

In another embodiment of the invention, the adenoviral vector cancomprise at least one nucleic acid sequence encoding a cytokine.“Cytokines” are known in the art as non-antibody proteins secreted byspecific cells (e.g., inflammatory leukocytes and some non-leukocyticcells), that act as intercellular mediators, such as by regulatingimmunity, inflammation, and hematopoiesis. Cytokines generally actlocally in a paracrine or autocrine rather than endocrine manner.Cytokines can be classified as a lymphokine (cytokines made bylymphocytes), a monokine (cytokines made by monocytes), a chemokine(cytokines with chemotactic activities), and an interleukin (cytokinesmade by one leukocyte and acting on other leukocytes). The cytokine canbe any suitable cytokine known in the art, including, but not limitedto, interferons, interleukins, RANTES, MCP-1, MIP-1α, and MIP-1β,granulocyte monocyte colony-stimulating factor (GM-CSF), and tumornecrosis factor (TNF) alpha. In a preferred embodiment, the cytokine isan interferon. In this regard, the adenoviral vector can comprise atleast one nucleic acid sequence encoding an interferon in addition tothe at least one nucleic acid sequence encoding an aphthovirus antigen.Alternatively, the adenoviral vector comprises at lease one nucleic acidsequence encoding an interferon and lacks a nucleic acid sequenceencoding an aphthovirus antigen. In either embodiment, the nucleic acidsequence can encode any suitable interferon (IFN). Suitable interferonsinclude, for example, Type I and Type II interferons. Type I interferonsinclude IFN-alpha, IFN-beta, IFN-delta, IFN-omega, and IFN-tau. Type IIinterferons include IFN-gamma. Interferons are a heterogeneous group ofproteins with some similar biological activities that are distinguishedfrom each other by many different physical and immunochemicalproperties. They are also encoded by different structural genes. Thehuman interferons IFN-beta and IFN-gamma are encoded by two differentsingle genes while human IFN-alpha constitutes a family of at least 23different genes. Most interferons are multifunctional proteins withbioactivities that are strictly species-specific. IFNs are synthesizedfollowing the activation of the immune system. In particular, IFNs aremainly known for their antiviral activities against a wide spectrum ofviruses. IFNs are synthesized, for example, by virus-infected cells andprotect other, non-infected but virus-sensitive cells against infection.In addition, interferons are also known to have protective effectsagainst some non-viral pathogens.

IFN-alpha and IFN-beta (IFN-alpha/beta) are known to have antiviralactivity and are the first line of host cell defense against virusinfection (see, e.g., Fields et al., eds., Virology, Lippincott-RavenPublishers, Philadelphia (1996)). Virus-infected cells are induced toexpress and secrete IFN alpha/beta, which binds to specific receptors onneighboring cells, priming them to a virus resistant state via a seriesof events leading to activation of IFN alpha/beta-stimulated genes(ISGs) (e.g., double-stranded (ds) RNA dependent protein kinase (PKR),2′-5′A synthetase/RNase L and Mx). The products of these genes affectviruses at different stages of their replication cycle, and differentviruses are susceptible to different ISG products. It has beendemonstrated that FMDV replication is highly sensitive to IFN-alpha or-beta, and that supernatant fluids containing porcine or bovineIFN-alpha/beta inhibit FMDV replication (see Chinsangaram et al., J.Virol., 73: 9891-9898 (1999)).

While in some embodiments of the invention the adenoviral vectorpreferably comprises one nucleic acid sequence encoding an aphthovirusantigen and/or one nucleic acid sequence encoding a cytokine, in otherembodiments of the invention the adenoviral vector can comprise two ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleic acid sequences,each of which encodes a different aphthovirus antigen, or a differentcytokine. In this respect, each of the two or more nucleic acidsequences preferably encodes a different aphthovirus antigen, adifferent cytokine, or combinations thereof. For example, the adenoviralvector can comprise two or more nucleic acid sequences, in which (i)each nucleic acid sequence encodes a different aphthovirus antigen, (ii)each nucleic acid encodes a different cytokine, (iii) one aphthovirusantigen-encoding nucleic acid sequence and two or more cytokine-encodingnucleic acid sequences, or (iv) one cytokine-encoding nucleic acidsequence and two or more aphthovirus antigen-encoding nucleic acidsequences.

It will be appreciated that an entire, intact viral, bacterial, orparasitic protein is not required to produce an immune response. Indeed,most antigenic epitopes are relatively small in size, and, therefore,protein fragments can be sufficient for exposure to the immune system ofthe mammal. In addition, a fusion protein can be generated between twoor more antigenic epitopes of one or more antigens. Delivery of fusionproteins via adenoviral vector to a mammal allows exposure of an immunesystem to multiple antigens and, accordingly, enables a single vaccinecomposition to provide immunity against multiple pathogens. In addition,the nucleic acid sequence encoding a particular antigen can be modifiedto enhance the recognition of the antigen by the mammalian host.

Each of the nucleic acid sequences in the inventive adenoviral vector isdesirably present as part of an expression cassette, i.e., a particularnucleotide sequence that possesses functions which facilitate subcloningand recovery of a nucleic acid sequence (e.g., one or more restrictionsites) or expression of a nucleic acid sequence (e.g., polyadenylationor splice sites). Each nucleic acid is preferably located in the E1region (e.g., replaces the E1 region in whole or in part) and/or the E4region of the adenoviral genome. For example, the E1 region can bereplaced by one or more promoter-variable expression cassettescomprising a nucleic acid. Alternatively, in embodiments where theadenoviral vector contains the E1 region but is deficient in the E4region, the E4 region can be replaced by one or more expressioncassettes. In this manner, inserting an expression cassette into the E4region of the adenoviral genome inhibits formation of “revertant E1adenovectors” (REA), because homologous recombination within the E1region and the E1 DNA of a complementing cell line (e.g., 293 cell) orhelper virus results in an E1-containing adenoviral genome that is toolarge for packaging inside an adenovirus capsid. Each expressioncassette can be inserted in a 3′-5′ orientation, e.g., oriented suchthat the direction of transcription of the expression cassette isopposite that of the surrounding adjacent adenoviral genome. However, itis also appropriate for an expression cassette to be inserted in a 5′-3′orientation with respect to the direction of transcription of thesurrounding genome. In this regard, it is possible for the inventiveadenoviral vector to comprise at least one nucleic acid sequence that isinserted into, for example, the E1 region in a 3′-5′ orientation, and atleast one nucleic acid sequence inserted into the E4 region in a 5′-3′orientation. The E1 and/or the E4 region can be replaced by two or moreexpression cassettes (e.g., a dual expression cassette). In thisembodiment, each of the expression cassettes can be positioned in anyorientation with respect to each other. For example, two expressioncassettes can be positioned such that each of the respective promotersis adjacent to the other. In this manner, one expression cassette is ina 5′-3′ orientation with respect to the direction of transcription ofthe adenoviral genome and the other expression cassette is in a 3′-5′orientation. By positioning two promoters adjacent to each other, theactivity of one of the promoters can be enhanced by the activity of theadjacent promoter.

In accordance with the invention, at least one nucleic acid sequence(e.g., one, two, three, or more nucleic acid sequences) is located inthe E1 region of the adenoviral genome, and at least one nucleic acidsequence (e.g., one, two, three, or more nucleic acid sequences) islocated in the E4 region of the adenoviral genome. While not preferred,all of the nucleic acid sequences can be located in either the E1 regionor the E4 region of the adenoviral genome. In embodiments where theadenoviral vector comprises two or more nucleic acid sequences encodingan aphthovirus antigen and/or a cytokine, each of the two or morenucleic acid sequences preferably are located in the E1 region or the E4region of the adenoviral genome. The insertion of an expression cassetteinto the adenoviral genome (e.g., into the E1 region of the genome) canbe facilitated by known methods, for example, by the introduction of aunique restriction site at a given position of the adenoviral genome. Asset forth above, preferably all or part of the E3 region of theadenoviral vector also is deleted.

Preferably, each nucleic acid sequence is operably linked to (i.e.,under the transcriptional control of) one or more promoter and/orenhancer elements, for example, as part of a promoter-variableexpression cassette. Techniques for operably linking sequences togetherare well known in the art. Any promoter or enhancer sequence can be usedin the context of the invention, so long as sufficient expression of thenucleic acid sequence is achieved and a robust immune response isgenerated. Preferably, the promoter is a heterologous promoter, in thatthe promoter is not obtained from, derived from, or based upon anaturally occurring promoter of the adenoviral vector. In this regard,the promoter can be a viral promoter. Suitable viral promoters include,for example, cytomegalovirus (CMV) promoters, such as the mouse CMVimmediate-early promoter (mCMV) or the human CMV immediate-earlypromoter (hCMV) (described in, for example, U.S. Pat. Nos. 5,168,062 and5,385,839), promoters derived from human immunodeficiency virus (HIV),such as the HIV long terminal repeat promoter, Rous sarcoma virus (RSV)promoters, such as the RSV long terminal repeat, mouse mammary tumorvirus (MMTV) promoters, HSV promoters, such as the Lap2 promoter or theherpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.,78: 144-145 (1981)), promoters derived from SV40 or Epstein Barr virus,an adenovirus promoter, such as the pIX promoter, an adeno-associatedviral promoter, such as the p5 promoter, and the like.

Alternatively, the promoter can be a cellular promoter, i.e., a promoterthat is native to eukaryotic, preferably animal, cells. In one aspect,the cellular promoter is a human cellular promoter or a bovine cellularpromoter. In another aspect, the cellular promoter is preferably aconstitutive promoter that works in a variety of cell types, such ascells associated with the immune system. Suitable constitutive promoterscan drive expression of genes encoding transcription factors,housekeeping genes, or structural genes common to eukaryotic cells.Suitable cellular promoters include, for example, a ubiquitin promoter(e.g., a UbC promoter) (see, e.g., Marinovic et al., J. Biol. Chem.,277(19): 16673-16681 (2002)), a human β-actin promoter, a chickenβ-actin promoter, an EGR promoter, an EF-1α promoter, a YY1 promoter, abasic leucine zipper nuclear factor-1 (BLZF-1) promoter, a neuronspecific enolase (NSE) promoter, a heat shock protein 70B (HSP70B)promoter, and a JEM-1 promoter.

Many of the above-described promoters are constitutive promoters.Instead of being a constitutive promoter, the promoter can be aninducible promoter, i.e., a promoter that is up- and/or down-regulatedin response to an appropriate signal. The use of a regulatable promoteror expression control sequence is particularly applicable to DNA vaccinedevelopment inasmuch as antigenic proteins, including viral and parasiteantigens, frequently are toxic to complementing cell lines. A promotercan be up-regulated by a radiant energy source or by a substance thatdistresses cells. For example, an expression control sequence can beup-regulated by drugs, hormones, ultrasound, light activated compounds,radiofrequency, chemotherapy, and cyofreezing. Thus, the promotersequence that regulates expression of the nucleic acid sequence encodingthe antigen and/or cytokine can contain at least one heterologousregulatory sequence responsive to regulation by an exogenous agent. Inone embodiment, the regulatory sequences operably linked to theantigen-encoding and/or cytokine-encoding nucleic acid sequences includecomponents of the tetracycline expression system, e.g., tet operatorsites. For instance, the antigen-encoding nucleic acid sequence isoperably linked to a promoter which is operably linked to one or moretet operator sites. When grown in complementing cells that express thetet repressor protein (tetR), the expression of the antigen-encoding orcytokine-encoding nucleic acid sequence is inhibited as a result of thetetR protein binding to the tetO sites. As a result, adenoviral vectorproduction proceeds without the interference of large or potentiallytoxic transgenes. Other suitable inducible promoter systems include, butare not limited to, the IL-8 promoter, the metallothionine induciblepromoter system, the bacterial lacZYA expression system, and the T7polymerase system. Further, promoters that are selectively activated atdifferent developmental stages (e.g., globin genes are differentiallytranscribed from globin-associated promoters in embryos and adults) canbe employed.

The promoter can be a tissue-specific promoter, i.e., a promoter that ispreferentially activated in a given tissue and results in expression ofa gene product in the tissue where activated. A tissue-specific promotersuitable for use in the invention can be chosen by the ordinarilyskilled artisan based upon the target tissue or cell-type. Preferredtissue-specific promoters for use in the inventive method are specificto immune cells, such as the dendritic-cell specific Dectin-2 promoterdescribed in Morita et al., Gene Ther., 8: 1729-37 (2001).

In yet another embodiment, the promoter can be a chimeric promoter. Apromoter is “chimeric” in that it comprises at least two nucleic acidsequence portions obtained from, derived from, or based upon at leasttwo different sources (e.g., two different regions of an organism'sgenome, two different organisms, or an organism combined with asynthetic sequence). Preferably, the two different nucleic acid sequenceportions exhibit less than about 40%, more preferably less than about25%, and even more preferably less than about 10% nucleic acid sequenceidentity to one another (which can be determined by methods describedelsewhere herein). Chimeric promoters can be generated using standardmolecular biology techniques, such as those described in Sambrook etal., supra, and Ausubel et al., supra.

A promoter can be selected for use in the invention by matching itsparticular pattern of activity with the desired pattern and level ofexpression of the antigen or cytokine. In embodiments where theadenoviral vector comprises two or more nucleic acid sequences encodingan antigen, the two or more antigen-encoding nucleic acid sequences areoperably linked to different promoters displaying distinct expressionprofiles. For example, a first promoter is selected to mediate aninitial peak of antigen production, thereby priming the immune systemagainst an encoded antigen. A second promoter is selected to driveproduction of the same or different antigen such that expression peaksseveral days after the initial peak of antigen production driven by thefirst promoter, thereby “boosting” the immune system against theantigen. Alternatively, a chimeric promoter can be constructed whichcombines the desirable aspects of multiple promoters. For example, aCMV-RSV hybrid promoter combining the CMV promoter's initial rush ofactivity with the RSV promoter's high maintenance level of activity isespecially preferred for use in many embodiments of the inventivemethod. In addition, a promoter can be modified to include heterologouselements that enhance its activity. For example, a human CMV promotersequence can include a synthetic splice signal, which enhancesexpression of a nucleic acid sequence operably linked thereto. In thatantigens can be toxic to eukaryotic cells, it may be advantageous tomodify the promoter to decrease activity in complementing cell linesused to propagate the adenoviral vector.

When the adenoviral vector comprises two or more nucleic acid sequences,the multiple nucleic acid sequences can be operably linked to the sameor different promoters. In a preferred embodiment of the invention, eachnucleic acid sequence is operably linked to a separate promoter. Whileit is preferred that each promoter is different, one or ordinary skillin the art will appreciate the advantages of using one particularlyefficient promoter to control expression of each nucleic acid sequencepresent in the adenoviral vector. Thus, each nucleic acid sequence canbe operably linked to the same promoter. In one aspect of the invention,the two or more nucleic acid sequences are operably linked to one ormore different promoters (e.g., two nucleic acid sequences are eachoperably linked to the same promoter, or each nucleic acid sequence isoperably linked to a different promoter). Most preferably, each of thetwo or more nucleic acid sequences is operably linked to a differentpromoter. The selection of an appropriate promoter for a given nucleicacid sequence will depend upon a number of factors, including promoterstrength and the position of the expression cassette within theadenoviral genome, and can be performed using routine methods known inthe art.

To optimize protein production, preferably the nucleic acid sequencefurther comprises a polyadenylation site 3′ of the coding sequence. Anysuitable polyadenylation sequence can be used, including a syntheticoptimized sequence, as well as the polyadenylation sequence of BGH(Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV(Epstein Barr Virus), and the papillomaviruses, including humanpapillomaviruses and BPV (Bovine Papilloma Virus). A preferredpolyadenylation sequence is the SV40 (Human Sarcoma Virus-40)polyadenylation sequence. Also, preferably all the proper transcriptionsignals (and translation signals, where appropriate) are correctlyarranged such that the nucleic acid sequence is properly expressed inthe cells into which it is introduced. If desired, the nucleic acidsequence also can incorporate splice sites (i.e., splice acceptor andsplice donor sites) to facilitate mRNA production.

The invention further provides a method of inducing an immune responseagainst an aphthovirus in a mammal, comprising administering to a mammalinfected by an aphthovirus a composition comprising the aforementionedadenoviral vector and a pharmaceutically acceptable carrier, wherein theaphthovirus antigen and/or cytokine are expressed in the mammal toinduce an immune response against the aphthovirus. Descriptions of theadenoviral vectors, aphthovirus antigen, and cytokine set forth above inconnection with other embodiments of the invention also are applicableto those same aspects of the aforesaid method.

In the method of the invention, the adenoviral vector preferably isadministered to a mammal, wherein the nucleic acid sequence(s) encodingthe aphthovirus antigen and/or cytokine is (are) expressed to induce animmune response against the aphthovirus. The mammal can be any suitablemammal, but preferably is a cloven-hooved animal. Suitable cloven-hoovedanimals include, for example, sheep, cattle, swine, goats, and deer. Theimmune response can be a humoral immune response, a cell-mediated immuneresponse, or, desirably, a combination of humoral and cell-mediatedimmunity. Ideally, the immune response provides protection uponsubsequent challenge with the infectious agent comprising the antigen.However, protective immunity is not required in the context of theinvention. The inventive method further can be used for antibodyproduction and harvesting.

Administering the adenoviral vector encoding the antigen and/or cytokinecan be one component of a multistep regimen for inducing an immuneresponse in a mammal. In particular, the inventive method can representone arm of a prime and boost immunization regimen. The inventive method,therefore, can comprise administering to the mammal a priming genetransfer vector comprising at least one nucleic acid sequence encodingan antigen and/or an cytokine prior to administering the adenoviralvector. The antigen and/or cytokine encoded by the priming gene transfervector can be the same or different from the antigen and/or cytokine ofthe adenoviral vector. The inventive adenoviral vector is thenadministered to boost the immune response to a given pathogen. More thanone boosting composition comprising the adenoviral vector can beprovided in any suitable timeframe (e.g., at least about 1 week, 2weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, or more following priming)to maintain immunity.

Any gene transfer vector can be employed as a priming gene transfervector, including, but not limited to, a plasmid, a retrovirus, anadeno-associated virus, a vaccinia virus, a herpesvirus, an alphavirus,or an adenovirus. Ideally, the priming gene transfer vector is a plasmidor an adenoviral vector. To maximize the effect of the priming regimen,the priming gene transfer vector can comprise more than one nucleic acidsequence encoding an antigen and/or a cytokine. Preferably, the priminggene transfer vector comprises two or more (e.g., 2, 3, 5, or more)nucleic acid sequences each encoding an antigen and/or a cytokine.Alternatively, an immune response can be primed or boosted byadministration of the antigen itself, e.g., an antigenic protein, thecytokine itself, intact pathogen (e.g., aphthovirus particles),inactivated pathogen, and the like.

Any route of administration can be used to deliver the adenoviral vectorto the mammal. Indeed, although more than one route can be used toadminister the adenoviral vector, a particular route can provide a moreimmediate and more effective reaction than another route. Preferably,the adenoviral vector is administered via intramuscular injection. Adose of adenoviral vector also can be applied or instilled into bodycavities, absorbed through the skin (e.g., via a transdermal patch),inhaled, ingested, topically applied to tissue, or administeredparenterally via, for instance, intravenous, peritoneal, orintraarterial administration.

The adenoviral vector can be administered in or on a device that allowscontrolled or sustained release, such as a sponge, biocompatiblemeshwork, mechanical reservoir, or mechanical implant. Implants (see,e.g., U.S. Pat. No. 5,443,505), devices (see, e.g., U.S. Pat. No.4,863,457), such as an implantable device, e.g., a mechanical reservoir,an implant, or a device comprised of a polymeric composition, areparticularly useful for administration of the adenoviral vector. Theadenoviral vector also can be administered in the form ofsustained-release formulations (see, e.g., U.S. Pat. No. 5,378,475)comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitinsulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate(BHET), and/or a polylactic-glycolic acid.

The dose of adenoviral vector administered to the mammal will depend ona number of factors, including the size of a target tissue, the extentof any side-effects, the particular route of administration, and thelike. The dose ideally comprises an “effective amount” of adenoviralvector, i.e., a dose of adenoviral vector which provokes a desiredimmune response in the mammal. The desired immune response can entailproduction of antibodies, protection upon subsequent challenge, immunetolerance, immune cell activation, and the like. Desirably, a singledose of adenoviral vector comprises at least about 1×10⁵ particles(which also is referred to as particle units) of the adenoviral vector.The dose preferably is at least about 1×10⁶ particles (e.g., about1×10⁶-1×10¹² particles), more preferably at least about 1×10⁷ particles,more preferably at least about 1×10⁸ particles (e.g., about 1×10⁸-1×10¹¹particles), and most preferably at least about 1×10⁹ particles (e.g.,about 1×10⁹-1×10¹⁰ particles) of the adenoviral vector. The dosedesirably comprises no more than about 1×10¹⁴ particles, preferably nomore than about 1×10¹³ particles, even more preferably no more thanabout 1×10¹² particles, even more preferably no more than about 1×10¹¹particles, and most preferably no more than about 1×10¹⁰ particles(e.g., no more than about 1×10⁹ particles). In other words, a singledose of adenoviral vector can comprise, for example, about 1×10⁶particle units (pu), 2×10⁶ pu, 4×10⁶ pu, 1×10⁷ pu, 2×10⁷ pu, 4×10⁷ pu,1×10⁸ pu, 2×10⁸ pu, 4×10⁸ pu, 5×10⁸ pu, 1×10⁹ pu, 2×10⁹ pu, 4×10⁹ pu,5×10⁹ pu, 1×10¹⁰ pu 2×10¹⁰ pu, 4×10¹⁰ pu, 1×10¹¹ pu, 2×10¹¹ pu, 4×10¹¹pu, 1×10¹² pu, 2×10¹² pu, or 4×10¹² pu of the adenoviral vector.

The adenoviral vector desirably is administered in a composition,preferably a pharmaceutically acceptable (e.g., physiologicallyacceptable) composition, which comprises a carrier, preferably apharmaceutically (e.g., physiologically acceptable) carrier and theadenoviral vector. Any suitable carrier can be used within the contextof the invention, and such carriers are well known in the art. Thechoice of carrier will be determined, in part, by the particular site towhich the composition is to be administered and the particular methodused to administer the composition. Ideally, in the context ofadenoviral vectors, the composition preferably is free ofreplication-competent adenovirus. The composition can optionally besterile or sterile with the exception of the inventive adenoviralvector.

Suitable formulations for the composition include aqueous andnon-aqueous solutions, isotonic sterile solutions, which can containanti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. The formulations canbe presented in unit-dose or multi-dose sealed containers, such asampules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, water, immediately prior to use. Extemporaneous solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Preferably, the carrier is a bufferedsaline solution. More preferably, the adenoviral vector for use in theinventive method is administered in a composition formulated to protectthe expression vector from damage prior to administration. For example,the composition can be formulated to reduce loss of the adenoviralvector on devices used to prepare, store, or administer the expressionvector, such as glassware, syringes, or needles. The composition can beformulated to decrease the light sensitivity and/or temperaturesensitivity of the expression vector. To this end, the compositionpreferably comprises a pharmaceutically acceptable liquid carrier, suchas, for example, those described above, and a stabilizing agent selectedfrom the group consisting of polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such acomposition will extend the shelf life of the vector, facilitateadministration, and increase the efficiency of the inventive method.Formulations for adenoviral vector-containing compositions are furtherdescribed in, for example, U.S. Pat. No. 6,225,289, U.S. Pat. No.6,514,943, U.S. Patent Application Publication 2003/0153065 A1, andInternational Patent Application Publication WO 00/34444. A compositionalso can be formulated to enhance transduction efficiency. In addition,one of ordinary skill in the art will appreciate that the adenoviralvector can be present in a composition with other therapeutic orbiologically-active agents. For example, factors that controlinflammation, such as ibuprofen or steroids, can be part of thecomposition to reduce swelling and inflammation associated with in vivoadministration of the viral vector. As discussed herein, immune systemstimulators or adjuvants, e.g., interleukins, lipopolysaccharide, anddouble-stranded RNA, can be administered to enhance or modify any immuneresponse to the antigen. Antibiotics, i.e., microbicides and fungicides,can be present to treat existing infection and/or reduce the risk offuture infection, such as infection associated with gene transferprocedures.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the production of an adenoviral vectorcomprising a nucleic acid sequence encoding an aphthovirus antigen.

An oligonucleotide containing two copies of the tet operator(5′-AGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGA GCT-3′) (SEQID NO: 4) was self-annealed, digested with SacI, and inserted at theSacI site between the TATA box and transcription start site of the CMVenhancer/promoter (GenBank X17403, nucleotides 174,314 to 173,566). Anartificial untranslated sequence (UTR) of 144 base pairs and 3′ splicesite sequences were inserted downstream of the CMV sequences, followedby a nucleic acid sequence encoding the A24 Cruzeiro FMDV empty capsidand a simian virus-40 (SV40) polyadenylation signal. The resulting A24empty capsid expression cassette was transferred to a shuttle plasmidcontaining adenovirus type 5 nucleotides 1-355 and 3333-5793 or3511-5793 flanking the expression cassette and a restriction site forlinearization.

Adenoviral vector genomes were constructed using the AdFast method (seeU.S. Pat. No. 6,329,200). Briefly, E. coli strain BJDE3 was transfectedwith 100 ng of shuttle plasmid containing the A24 empty capsidexpression cassette and 100 ng of a GV.11 base plasmid. The desiredrecombinant plasmids, containing deletions in the E1, E3, and E4 regionsof the adenoviral genome and the expression cassette were identified byrestriction digestion of DNA from individual bacterial colonies. Theplasmids were further purified by transformation of recombinationnegative DH5α E. coli and single-colony isolation by standardmicrobiological methods. Isolation of a single genetic clone of thefinal vector genome was achieved by two sequential colony-growth stepsin bacteria. The adenoviral vector plasmid structures were confirmed byrestriction digestion analysis and DNA sequencing.

A 293-ORF6 cell line (Brough et al., J. Virol., 70, 6497-6501 (1996))stably expressing the TetR protein (293-ORF6TetR) was generated bytransfecting 293-ORF6 cells with 2 μg of a HpaI-linearized pRSVTetR.hygplasmid. After 24 hours the cells were split to ten 10 cm dishes andincubated in 250 μg/ml hygromycin.

293-ORF6TetR cells were transduced with the above-described E1-, E3-,E4-deleted adenoviral vectors comprising the nucleic acid sequenceencoding the A24 FMDV empty capsid expressed under the control of aCMV-tetO promoter (A24 GV11).

Two research lots of the A24 GV11 vector were evaluated for thedevelopment of a serum antibody response against FMDV over time. Onevector lot was produced and expanded in the 293-ORF6TetR cells, whilethe other was produced and expanded in 293-ORF6 cells. Cows wereadministered 5×10⁹ pfu of each vector lot. At 4, 7, 14, and 21 days postinoculation, serum was obtained from treated cows, and anti-FMDVantibody responses were measured using methods known in the art. Bothvector lots produced a significant anti-FMDV antibody titer.

The results of this example demonstrate that an E1/E4-deficientadenoviral vector comprising a nucleic acid sequence encoding an FMDVantigen elicits an antibody response in vivo.

EXAMPLE 2

This example demonstrates that an adenoviral vector encoding an FMDVantigen induces protection against FMDV challenge in cows.

A dose of 5×10⁸ particle forming units (pfu) or 5×10⁹ pfu of the A24GV11 described in Example 1 was administered intramuscularly to cows (6cows per dose) on “day 1”. On day 7, cows were challenged with 2×10⁴infectious units directly injected into the tongue of vaccinated cows.Challenged cows were then evaluated for FMDV-induced lesions on theirfeet, as well as viremia.

In addition to challenge with direct inoculation of FMDV, a second groupof six vaccinated cows were subject to contact challenge. Specifically,a dose of 5×10⁹ pfu of the A24 GV11 vector described in Example 1 wasadministered intramuscularly to cows (6 cows per dose) on “day 1.” Sevendays post vaccination, cows were placed in contact (i.e., in the sameroom) with cows infected with FMDV.

Cows subject to direct inoculation challenge showed no systemic clinicaldisease, and no viremia occurred in all 12 vaccinated animals. Five ofsix vaccinated cows subject to contact challenge showed no systemicclinical disease. Only one of these six animals developed a tonguelesion. None of the contact-challenged cows developed viremia.

This example demonstrates a method of inducing a protective immuneresponse against FMDV using the inventive adenoviral vector.

EXAMPLE 3

This example demonstrates that an adenoviral vector encoding an FMDVantigen induces protection against FMDV challenge in cows.

Four groups of 7 or 6 cows each were administered one of the followingdoses of the A24 GV11 adenoviral vector described in Example 1: GroupA—5×10⁹ focus forming units (ffu), Group B—1×10⁸ ffu, Group C—5×10⁶ ffu,Group D—no adenovirus (control). Seven days after immunization, cows ineach group were challenged with 1×10⁶ infectious units of A24 FMDVdirectly injected into the tongue of vaccinated cows. Challenged cowswere then evaluated for FMDV-induced fever, virus neutralizing antibodytiter, virus isolation (positive or negative), and generalization ofinfection (e.g., presence, location, and number of lesions). The resultsof this experiment are set forth in FIGS. 1A-1D. Neutralizing antibodytiters raised against FMDV serotype A24 and serotype 5 adenovirus alsowere measured in challenged cows using methods known in the art. Theresults of these experiments are set forth in FIGS. 2A and 2B.

Of the doses tested, the 5×10⁹ ffu dose of A24 GV11 was most effectiveat inducing a protective immune response against FMDV challenge incattle. In addition, vaccination with A24 GV11 elicited a neutralizingantibody response against the A24 FMDV, but did not elicit a significantneutralizing antibody response against the adenoviral vector backbone.

The results of this example demonstrate the effectiveness of a method ofinducing a protective immune response against FMDV using the inventiveadenoviral vector.

EXAMPLE 4

This example demonstrates that an adenoviral vector encoding an FMDVantigen induces protection against FMDV challenge in cows as well as aninactivated FMDV vaccine.

Five groups of cows each were administered a dose of the A24 GV11adenoviral vector described in Example 1 or a dose of inactivated A24FMDV according to the dosing schedule in Table 1.

TABLE 1 Day of Number Challenge of Cows post FMDV Group (n) Vaccine DoseVaccination Challenge (ID₅₀) A 5 A24 GV11 2 × 10⁹ FFU 7 1 × 10⁶ B 5 A24GV11 2 × 10⁹ FFU 4 1 × 10⁶ C 5 Inactivated A24 2 mL 7 1 × 10⁶ D 5Inactivated A24 2 mL 4 1 × 10⁶ E 6 None N/A N/A 1 × 10⁶

At 4 days or 7 days after vaccination, vaccinated cows in each groupwere challenged with 1×10⁶ infectious units of A24 FMDV directlyinjected into the tongue of vaccinated cows. Challenged cows were thenevaluated for neutralizing antibody titers directed against A24 FMDV andserotype 5 adenovirus. The results of this study are set forth in FIGS.3A and 3B.

Vaccination with A24 GV11 and inactivated A24 FMDV elicited similarneutralizing antibody responses against the A24 FMDV, but did not elicita significant neutralizing antibody response against the adenoviralvector backbone. The results of this example demonstrate that theinventive adenoviral vector is as effective in generating an immuneresponse against FMDV as is inactivated FMDV.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. An adenoviral vector comprising an adenoviral genome and at least onenucleic acid sequence encoding an Aphthovirus empty virus capsid andoptionally a cytokine operably linked to a promoter, wherein (i) thepromoter consists of (a) SEQ ID NO:4, (b) a CMV enhancer/promotersequence, (c) an untranslated region (UTR), and (d) one or more 3′splice sequences, and (ii) the adenoviral vector isreplication-deficient and requires complementation of only the E1region, only the E4 region, or only both the E1 and E4 regions of theadenoviral genome for propagation.
 2. The adenoviral vector of claim 1,wherein the adenoviral vector comprises a first nucleic acid sequenceencoding an Aphthovirus empty virus capsid and a second nucleic acidsequence encoding a cytokine, wherein the first and second nucleic acidsequences are different.
 3. The adenoviral vector of claim 2, whereinthe cytokine is an interferon.
 4. The adenoviral vector of claim 3,wherein the interferon is selected from the group consisting ofinterferon alpha, interferon beta, and interferon gamma.
 5. Theadenoviral vector of claim 1, wherein the Aphthovirus is afoot-and-mouth disease virus (FMDV).
 6. The adenoviral vector of claim5, wherein the FMDV is of a serotype selected from the group consistingof A, 0, C, Asia 1, South African Territories (SAT) 1, SAT 2, and SAT 3.7. The adenoviral vector of claim 5, wherein the FMDV is of strain A24Cruzeiro.
 8. The adenoviral vector of claim 1, wherein the adenoviralgenome lacks the entire E1 region and at least a portion of the E4region.
 9. The adenoviral vector of claim 1, wherein the adenoviralgenome lacks at least a portion of the E3 region.